CN111808954B - lncRNA and application thereof in diseases - Google Patents
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- CN111808954B CN111808954B CN202010515230.7A CN202010515230A CN111808954B CN 111808954 B CN111808954 B CN 111808954B CN 202010515230 A CN202010515230 A CN 202010515230A CN 111808954 B CN111808954 B CN 111808954B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q2600/158—Expression markers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses lncRNA and application thereof in diseases, belonging to the field of biomedicine, wherein the lncRNA comprises RP11-830F9.5. By detecting lncRNA, the risk of the liver cancer of the subject can be evaluated, and auxiliary reference is provided for diagnosis of the liver cancer; by changing the expression level of lncRNA, the proliferation, migration and invasion of liver cancer cells can be influenced, and a molecular target is provided for the treatment of liver cancer.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to lncRNA and application thereof in diseases.
Background
Liver cancer (Liver cancer) refers to a malignant tumor that occurs in or begins from the Liver, and symptoms of Liver cancer include mass or pain under the right side of the costal cage, ascites, jaundice, bruising easily, weight loss, and physical weakness. Liver cancer is one of the most mortality malignant tumors. It is the fifth most common malignancy in the world and also the third leading cause of cancer death. More than 50 million people worldwide suffer from liver cancer each year, of which over half are dying from liver cancer in China, about 38.3 million people annually, accounting for 51% of the number of worldwide deaths from liver cancer, placing a heavy burden on our society and medicine (Sia D, villanuva a, et al. Liver cancer cell of origin, molecular class, and effects on patient diagnosis [ J ]. Gastroenterology,2017, 152. Hepatocellular carcinoma (HCC) occurs in the context of chronic liver inflammation and is highly correlated with chronic viral hepatitis infections (hepatitis b or c) or with exposure to toxins such as alcohol or aflatoxins. China is a high-incidence country of hepatitis B, and the situation of hepatocellular carcinoma is thus controlled to be particularly severe (Parikh ND, fu S et al. Risk assessment of hepatocellular pathogens with lipids c in the chip and the USA [ J ] diagnostic diseases and sciences,2017, 62. Many patients are staged to the middle or late stage at the time of initial diagnosis, and some have missed the best opportunity for diagnosis and treatment, resulting in rather poor prognosis (Zhu ZX, huang J et al. Treatment protocol for a procedure in medicine: radio frequency abnormality treatment [ J ]. Japanese patent J outer of clinical oncology,2016, 46. In recent years, the development of deep sequencing and DNA tiling arrays has drastically changed our opinion on gene organization and content: it is estimated that up to 70% of the genome is transcribed, but only 2% of the human genome is ultimately translated into protein (Polychronopoulos D, king JWD et al. Conserved non-coding elements: development gene regulation genome organization [ J ]2017,45, 12611-12624.). With the development of research, long non-coding RNA (1 ncRNA), a type of transcript longer than 200 nucleotides and not coding for protein, is discovered by researchers. The 1ncRNA is further classified into 5 classes based on its relative position in the genome to the gene encoding the protein, namely sense 1ncRNA, antisense 1ncRNA, bidirectional 1ncRNA, intragenic 1ncRNA and intergenic 1ncRNA (Jarroux J, morillon Aet. History, discovery, and classification of lncernas [ J ] Advances in experimental media and biology,2017,1008 1-46.. Currently, 1ncRNA has been proved to play a role in various biological processes, such as regulation in growth and development, participation in stabilizing immune system and the like, and is more relevant to the occurrence and development of tumors. LncRNAs are aberrantly expressed in various cancers and are involved in various cancer biological processes. For example, 1 ncRNAALAT 1 is found to be abnormally expressed in early stage small cell lung Cancer and can be used as one of prognostic indicators showing metastatic potential (Li ZX, zhu QN, et al. Maltl: apoptosis biology in Cancer [ J ]. Cancer management and research,2018, 10. The relationship of 1ncRNA to metastasis in various cancers was subsequently revealed by various subject groups, and was confirmed to be closely related to the development and metastasis of many tumors. Therefore, more long-chain non-coding RNA with important regulation and control effects on tumor metastasis is found to have non-negligible effects on recognizing the occurrence and development of tumors and providing corresponding prevention and treatment measures.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention finds lncRNA with obvious expression difference by detecting the expression of lncRNA in a liver cancer sample in a tumor tissue and a normal tissue through extensive and intensive research, and discusses the relationship between the lncRNA and the occurrence of liver cancer, thereby finding a better way and a better method for diagnosis and targeted therapy of the liver cancer.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a genetic marker for liver cancer, said marker comprising RP11-830f9.5.
In a second aspect, the present invention provides a reagent for detecting a gene marker according to the first aspect of the present invention.
In a third aspect, the invention provides a use of a gene marker according to the first aspect of the invention or an agent according to the second aspect of the invention, the use comprising:
1) The method is used for constructing a calculation model for predicting the risk of the liver cancer;
2) Preparing a reagent for diagnosing liver cancer; or
3) Preparing a liver cancer diagnosis kit.
Further, the input variable of the calculation model is the expression level of the gene marker according to the first aspect of the present invention.
Further, the method for measuring the expression level of the gene marker includes any one or more of reverse transcription PCR, real-time quantitative PCR, in situ hybridization, and a chip technology.
In a fourth aspect, the present invention provides a kit for diagnosing liver cancer, which comprises the reagent according to the second aspect of the present invention.
Further, the reagent includes a probe that specifically recognizes RP11-830F9.5 or a primer that specifically amplifies RP11-830F9.5.
Furthermore, the primer sequence of the specific amplification RP11-830F9.5 gene is shown as SEQ ID NO. 1-2.
The fifth aspect of the invention provides an application of an RP11-830F9.5 gene or an accelerant thereof in preparing a pharmaceutical composition for preventing and/or treating liver cancer.
Further, the promoter comprises an RP11-830F9.5 gene expression product, a promoting miRNA, a promoting transcription regulatory factor or a promoting targeting small molecule compound.
Further, the promoter is an RP11-830F9.5 gene expression product or a construct for producing the RP11-830F9.5 gene expression product.
The sixth aspect of the invention provides a pharmaceutical composition for treating liver cancer, which comprises an accelerator of RP11-830F9.5.
Further, the promoter is an RP11-830F9.5 gene expression product or a construct for producing the RP11-830F9.5 gene expression product.
Detailed Description
The inventor of the invention finds that the expression of RP11-830F9.5 is obviously reduced in liver cancer tissues through a large amount of experiments and repeated research, and detects the functional influence of the gene on liver cancer cells by increasing the expression of the RP11-830F9.5 gene in order to discuss the correlation between the RP11-830F9.5 and the occurrence and development of liver cancer.
In the present invention, the term "RP11-830F9.5" is located on chromosome 16 and includes the RP11-830F9.5 gene and its homologues, mutations, and isoforms. The term encompasses full-length, unprocessed RP11-830F9.5, as well as any form of RP11-830F9.5 that results from processing in cells. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RP11-830f9.5. Four transcripts exist at present in RP11-830F9.5, and the sequences are respectively ENST00000565053.1, ENST00000569249.1, ENST00000562574.1 and ENST00000562405.1. A representative RP11-830F9.5 sequence is shown in ENST 00000565053.1.
The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded.
The invention also relates to nucleic acid fragments hybridizing to the sequences described above, including sense and antisense nucleic acid fragments. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragment can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate a polynucleotide of RP11-830F9.5.
The full-length human RP11-830F9.5 nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together.
A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
Methods well known to those skilled in the art can be used to construct expression vectors containing the human RP11-830F9.5 coding DNA sequence and appropriate transcription control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct RNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.
Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl 2 Methods, the steps used are well known in the art. Another approach is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The promoter of RP11-830F9.5 of the invention can promote the expression of RP11-830F9.5 when being applied (dosed) on treatment, thereby inhibiting liver cancer. In a preferred embodiment, the RP11-830F9.5 promoter comprises an RP11-830F9.59 gene expression product, a promoting miRNA, a promoting transcription regulatory factor or a promoting targeting small molecule compound.
Typically, these enhancers will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5 to about 8, preferably a pH of about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
The invention also provides a pharmaceutical composition, which contains a safe and effective amount of the KLF9 protein or the promoter thereof and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.
In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to: alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The subject in need of treatment includes those already suffering from the condition or disorder as well as those predisposed to suffering from the condition or disorder, or those in which the condition or disorder is to be prevented.
The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. Where the drug can prevent the growth of and/or kill existing cancer cells, the drug may be cytostatic and/or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rates (RR).
Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited.
In the present invention, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid linked thereto. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
The invention has the advantages and beneficial effects that:
the invention discovers the characteristic gene of the liver cancer for the first time, establishes a calculation model for predicting the liver cancer, a product for diagnosing the liver cancer and a pharmaceutical composition for treating the liver cancer, and has higher sensitivity and specificity when RP11-830F9.5 is used as a detection variable for diagnosis.
Drawings
FIG. 1 is a graph showing the expression of RP11-830F9.5 in a sample.
FIG. 2 is a graph of the diagnostic performance of RP11-830F9.5.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1 detection of expression of RP11-830F9.5 in hepatocellular carcinoma
1. Sample collection
The cancer tissues of 27 patients with primary hepatocellular carcinoma and corresponding paracancerous tissue samples (liver tissues with the distance from the tumor edge being more than or equal to 5 cm) were collected, and the matched cancer tissues were pathologically confirmed to be hepatocellular carcinoma. The specimen is cleaned by a large amount of normal saline in the operation, then is immediately subpackaged, is frozen by liquid nitrogen and is put into a refrigerator at minus 80 ℃ for freezing storage.
2. Preparation and Mass analysis of RNA samples
Extraction of tissue total RNA Using TRIZOL method
1) Shearing tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1min; standing at room temperature for 10min to completely decompose nucleoprotein.
2) Adding 200 μ l chloroform (chloroform), covering the tube, shaking vigorously for 15s, and standing at room temperature for 10min.
3) Centrifuge at 11000rpm for 15min at 4 ℃.
4) Transferring the water sample layer into a new centrifuge tube, and adding 500 mu l of isopropanol; after the mixture was inverted and mixed, the mixture was left standing at room temperature for 10min.
5) Centrifuge at 11000rpm for 15min at 4 ℃.
6) The liquid was carefully aspirated off with a gun, the precipitate was left at the bottom of the tube, 1ml of 75% ethanol was added, the mixture was shaken on a shaker for 5s, and the precipitate was washed once.
7) Centrifuge at 8000rpm for 5min at 4 ℃.
8) Carefully removing supernatant, drying the precipitate for 10min, and dissolving the precipitate with appropriate amount of water for 10min.
9) And detecting the concentration of the RNA, and identifying the yield and purity of the RNA.
3. QPCR detection
1) Primer design
Primers were designed based on the gene sequences of RP11-830F9.5 and GADPH, and the specific primer sequences were as follows:
RP11-830F9.5 gene:
the forward primer is 5 'GAACACACGCTGGCTCTGAG-3' (SEQ ID NO. 1);
the reverse primer is 5 'AACCTTAGACGCTGTATTGAA-3' (SEQ ID NO. 2).
GAPDH gene:
the forward primer is 5;
the reverse primer is 5.
2) Reverse transcription reaction
Using FastQ μ ant cDNA first strand synthesis kit (cat # KR 106) to perform IncRNA reverse transcription, first, genomic DNA reaction was removed, 5 XgDNAB μ ffer 2.0 μ l, total RNA1 μ g, and RNase Free ddH were added to a test tube 2 O to make the total volume 10. Mu.l, heating in a water bath at 42 ℃ for 3min, and adding 10 Xfast RT B. Mu.ffer 2.0. Mu.l, RT Enzyme Mix 1.0. Mu.l, FQ-RT Primer Mix 2.0. Mu.l, RNase Free ddH 2 O5.0. Mu.l, mixing, adding into the test tube, mixing to give 20. Mu.l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3min.
3) QPCR amplification assay
Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP 205) and the experimental procedures were performed according to the product instructions.
A20. Mu.l reaction was used: 2 XSuperReal PreMix Plus 10. Mu.l, forward and reverse primers (10. Mu.M) 0.6. Mu.l each, 5 XROX Reference Dye △ 2. Mu.l, DNA template 2. Mu.l, sterilized distilled water 4.8. Mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.
The amplification procedure was: 95 ℃ 15min, (95 ℃ 10s,55 ℃ 30s,72 ℃ 32 s). Times.40 cycles.
4) Screening for cDNA template concentration
Mixing cDNA of each sample, diluting the cDNA by 10 times gradient (10, 100, 1000, 10000 and 100000 times) by taking the cDNA as a template, taking 2 mu l of each diluted sample as the template, respectively amplifying by using a target gene primer and an internal reference gene primer, simultaneously carrying out melting curve analysis at 60-95 ℃, and screening the concentration of the template according to the principle of high amplification efficiency and single peak of the melting curve.
From the dissolution curve, it can be seen that when 10-fold dilution of cDNA was performed, the amplification efficiency of PCR was high and the single peak of the dissolution curve was good.
5) Sample Real Time PCR detection
After 10-fold dilution of cDNA of each sample, 2 μ l of cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. Performing melting curve analysis at 60-95 deg.C, and determining target band by melting curve analysis and electrophoresis, 2 -ΔΔCT The method is used for relative quantification.
4. As a result, the
The QPCR results showed that, with the expression level of RP11-830f9.5 in the control group (tissue near cancer) as the reference 1, the expression of RP11-830f9.5 was down-regulated in hepatocellular carcinoma tissue compared to the tissue near cancer (fig. 1), and the difference was statistically significant (P < 0.05).
The specific expression is shown in table 1, and there are 25 samples showing significant down-regulation of RP11-830f9.5, 21 cancer tissue samples and 4 cancer-adjacent tissue samples. The RP11-830F9.5 has higher specificity and sensitivity when applied to the diagnosis of hepatocellular carcinoma.
TABLE 1 Positive status of genes in diseases
Example 2 detection of diagnostic efficacy of RP11-830F9.5
1. Data collection
The expression profile data of lncRNA were downloaded from TCGA database, which included 371 liver cancer tissues and 50 paracarcinoma tissues.
2. ROC curve analysis
Using pROC package in R language, the subject working characteristics of lncRNA RP11-830F9.5 were analyzed, two accurate confidence spaces were calculated, and ROC curves were plotted.
3. Results
The ROC analysis results are shown in fig. 2, and it can be seen that RP11-830f9.5 has a high area under the curve (AUC = 0.899) as a detection variable, the optimal critical point is 23.500, the specificity of the optimal critical point is 0.980, and the sensitivity is 0.768. It is demonstrated that the diagnosis of hepatocellular carcinoma using RP11-830F9.5 has higher sensitivity and specificity.
Example 3 functional verification of RP11-830F9.5
1. Cell culture
The human hepatoma cell line HepG2 was purchased from the Shanghai cell bank and the cell lines were each determined at 37 ℃ and 5% CO in DMEM medium containing 10% fetal bovine serum and 1% P/S 2 Cultured in an incubator. Cell growth was observed daily and fluid changed every other day.
2. Transfection
2.1 construction of overexpression vectors
An overexpression vector of RP11-830F9.5 is designed and synthesized by the Shanghai Jikai gene, and the experiment is divided into three groups: control group (HepG 2), negative control group (transfection empty) and experimental group (overexpression RP 11-830F9.5)
2.2 transfection
Transfection was performed according to the instructions of the lipofectamine 2000 transfection reagent from Invitrogen. The method comprises the following specific steps:
1) Adding the hepatoma cell line HepG2 to the DMEM medium containing 10% fetal bovine serum and then incubating at 37 ℃ with 5% CO 2 Cultured in an incubator. When the cells grew to confluence at 80-90%, the serum fraction in the cell flasks was removed by washing 3 times with PBS, and 1-2mL of the serum fraction was addedDigestion solution containing 0.25% pancreatin 0.02% EDTA, digestion was terminated by adding 1mL of complete medium when the cells became single cells.
2) Transferring the cells into a centrifuge tube, placing into a normal temperature centrifuge, centrifuging at 1000g/min for 5min to collect cell precipitate, and resuspending the cells to 1 × 10 with complete culture medium 5 Per mL, pipetting 500. Mu.L, inoculating into 24-well plate, placing at 37 deg.C, 5% 2 Was cultured overnight in an incubator.
3) Lipofectamine 2000 was added to the OPTI-MEM culture medium, and the mixture was incubated at room temperature for 5min.
4) The over-expression vector is added into the OPTI-MEM culture solution and mixed evenly.
5) Mixing the diluted 3) and 4) uniformly, and standing at room temperature for 20min.
6) Adding the mixture into 24-well plate of serum-free medium-cultured cells, shaking gently, mixing, adding 37 deg.C and 5% CO 2 After culturing for 6h in the incubator, the cell culture solution is replaced by fresh complete culture medium for continuous culture.
3. QPCR detection of the expression level of RP11-830F9.5 in cells
After 48h of transfection and culture of each group of cells, total RNA of the cells was extracted by Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method of example 1.
4. CCK-8 method for detecting cell proliferation capacity
1) After 24h of transfection of each group of cells, the cells were washed 3 times with PBS to remove serum components inside the cells, then the cells were sufficiently digested with trypsin, centrifuged at 1000rpm for 5min to collect the cells, the supernatant was discarded and resuspended in 1ml of DMEM medium containing 10% fetal bovine serum, and the cells were counted by a cell counting plate.
2) Cells were diluted to 1X 10 with DMEM medium containing 10% fetal bovine serum 4 mL, then add 200 μ L cells to each well of a 96-well plate (5 duplicate wells were seeded with each cell).
3) To the other wells, which were not plated with cells around the 96-well plate, 200. Mu.L of PBS was added.
4) Placing the 96-well plate inoculated with tumor cells at 37 ℃ and 5% 2 After culturing for 72h in the cell culture box, 10 mu L of CCK-8, placing the mixture into an incubator to incubate for 1h.
5) The plate was removed and absorbance at a wavelength of 450nm was measured using a microplate reader.
5. Cell migration assay
1) 24h after transfection of each group of cells, the cells were washed 3 times with PBS to remove serum components from the cells, then the cells were digested with trypsin, the cell suspension was transferred to a 15mL centrifuge tube, and then placed in a room temperature centrifuge and centrifuged at 1000rpm for 5min to collect the cells. The supernatant was discarded and the cells were resuspended in 1ml of DMEM medium without fetal bovine serum and counted using a cell counting plate. Cells were diluted to 1X 10 with DMEM medium without fetal bovine serum 6 Per mL, suction 1X 10 5 One cell was added dropwise to the upper chamber of the Transwell chamber.
2) Add 600. Mu.L of complete medium containing 10% serum to the lower chamber of the Transwell chamber, then place the upper chamber into the well plate, at 37 ℃ and 5% CO 2 The cells are cultured in the cell culture box for 24 hours.
3) The chamber was removed, the medium in the chamber was gently discarded, washed 2 times with PBS, and fixed in 75% ethanol fixative for 15min.
4) Cells in the upper chamber of the Transwell were gently wiped off with a cotton swab and the chamber was stained in crystal violet for 15min.
5) The chamber was removed, washed 3-5 times with PBS to remove excess cell stain, and then observed under a microscope.
6. Cell invasion assay
The matrigel was diluted with serum-free medium 1. The remaining steps were the same as the cell migration experiment.
7. Statistical analysis
All data were obtained from three independent experiments, shown by Mean SD. Differences between groups were analyzed using paired sample t-test, with p <0.05 indicating that the differences were statistically different.
8. Results
1) The transfection results showed that the expression level (5.38 ± 0.707) of RP11-830f9.5 was significantly increased after transfection of the vector overexpressing RP11-830f9.5 in the experimental group compared to the control group (P value =0.0086, x) when the expression level (0.94 ± 0.0265) of RP11-830f9.5 in the unloaded negative control group was determined as 1 based on the expression level of RP11-830f9.5 in the control group (HepG 2) (vs negative control group in the control group, P value =0.0086, x) and was not significantly changed when RP11-830f9.5 in the unloaded negative control group was transfected (P value =0.0591, ns)
2) The cell proliferation test result shows that the cell proliferation activity of the experimental group is remarkably reduced (0.67 +/-0.0765) compared with the cell proliferation activity of the negative control group (1.25 +/-0.0935) (the negative control group vs experimental group, P =0.0004, x), which indicates that RP11-830F9.5 influences the proliferation of the hepatoma cells, and indicates that RP11-830F9.5 can be used as a molecular target for treating the hepatoma.
3) Cell migration and invasion experimental results show that compared with a negative control group (184.7 +/-10.21) of a migration experiment, the cell transmembrane number of the experimental group of the migration experiment is remarkably reduced (95 +/-6.557), and the difference has statistical significance (a negative control group vs experimental group, P = 0.0108), which indicates that the increase of the expression level of RP11-830F9.5 can reduce the migration capacity of cells, and indicates that RP11-830F9.5 can be applied to the treatment of liver cancer metastasis; compared with a negative control group (134.7 +/-9.074) of an invasive experiment, the cell transmembrane number of the experimental group of the invasive experiment is obviously reduced (68.33 +/-10.02), and the difference has statistical significance (a negative control group vs experimental group, P = 0.0259), which indicates that the increase of the expression level of RP11-830F9.5 can reduce the invasive capacity of cells, and indicates that RP11-830F9.5 can be applied to the treatment of liver cancer infiltration.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.
Sequence listing
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Shandong first medical university (Shandong provincial academy of medicine science)
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Claims (7)
1. Use of a reagent for detecting the expression level of a gene marker RP11-830f9.5, comprising:
1) Preparing a reagent for diagnosing liver cancer; or
2) Preparing a liver cancer diagnosis kit.
2. The use according to claim 1, wherein the method for determining the expression level of the gene marker comprises any one or more of reverse transcription PCR, real-time quantitative PCR, in situ hybridization, and chip technology.
3. The use according to claim 1, wherein the reagent comprises a probe that specifically recognizes RP11-830f9.5 or a primer that specifically amplifies RP11-830f9.5.
4. The use according to claim 3, wherein the primer sequence for specifically amplifying the RP11-830F9.5 gene is shown as SEQ ID No. 1-2.
5. An application of RP11-830F9.5 gene promoter in preparation of medicine composition for curing liver cancer.
6. The use of claim 5, wherein the promoter comprises an RP11-830f9.5 gene expression product, a promoting miRNA, a promoting transcription regulatory factor, or a promoting targeted small molecule compound.
7. Use according to claim 6, wherein the promoter is an RP11-830f9.5 gene expression product or a construct producing an RP11-830f9.5 gene expression product.
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2020
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